Industrial fermentation predominately uses glucose as a feedstock for the production of a multitude of proteins, enzymes, amino acids, alcohols, organic acids, pharmaceuticals and other chemicals. In many applications, the glucose is produced from the enzymatic conversion of carbon substrates such as biomass and starch. Starch, which is abundantly found in green plants, accumulates as microscopic granules varying in diameter from 0.5 to 175 microns. The partial crystalline nature of these starch granules imparts insolubility in cold water. As a result, the solubilization of starch granules in water requires a tremendous amount of heat energy to disrupt the crystalline structure of the granule resulting in the solubilization of partially hydrolyzed starch. Numerous solubilization processes have been employed and these include direct and indirect heating systems, such as direct heating by steam injection. (See for example, STARCH CHEMISTRY AND TECHNOLOGY, eds R. L. Whistler et al., 2nd Ed., 1984 Academic Press Inc., Orlando, Fla.; STARCH CONVERSION TECHNOLOGY, Eds. G. M. A. Van Beynum et al., Food Science and Technology Series, Marcel Dekker Inc., NY; and THE ALCOHOL TEXTBOOK, 3rd Ed., Eds. K. Jacques, T. P. Lyons and D. R. Kelsall, 1999, Nottingham University Press, UK).
In general, two enzyme steps have been used for the hydrolysis of starch to glucose. The first step is a liquefaction step, and the second step is a saccharification step. In the liquefaction step, the insoluble starch granules are slurried in water, gelatinized with heat and hydrolyzed by a thermostable alpha amylase (EC.3.2.1.1, alpha (1-4)-glucan glucanohydrolase) in the presence of added calcium to produce a mash of dextrins. The resulting mash is generally cooled to about 60 to 65° C. In the saccharification step, the soluble dextrins (sugars) are further hydrolyzed to dextrose (glucose) by an enzyme having glucoamylase (EC 3.2.1.3, alpha (1,4)-glucan glucohydrolase) activity. Glucose may then be used as an end product or used as a precursor to be converted into other commercially important desired end products, such as fructose, sorbitol, ethanol, lactic acid, ascorbic acid (ASA) intermediates and 1,3 propanediol.
In the late 1950s, glucoamylases derived from Aspergillus niger were commercialized, and these enzymes significantly improved the conversion of starch to glucose. Another significant improvement occurred in the 1970s. A thermostable alpha amylase having improved thermostability, pH stability and lower calcium dependency was derived and commercialized from Bacillus lichenifonnis (U.S. Pat. No. 3,912,590).
Further industrial processes have been adopted by the starch sweetener industry for the enzyme liquefaction process (U.S. Pat. No. 5,322,778). Some of these processes include, a low temperature process (105-110° C. for 5-8 min) with lower steam requirements and a high temperature process (148° C.+/−5° C. for 8-10 sec), which improves gelatinization of the starch granules resulting in improved filtration characteristics and quality of the liquefied starch substrate (Shetty, et al., (1988) Cereal Foods World 33:929-934).
While enzyme starch liquefaction processes are well established, improvements with respect to yield loss, processing costs, energy consumption, pH adjustments, temperature thresholds, calcium requirement and the levels of retrograded starch would be desirable. In particular, it is well known that residual alpha amylase from the liquefaction step, under saccharification conditions, has an adverse effect on process efficiency and that the residual alpha amylase must be inactivated prior to saccharification by glucoamylases. Inactivation is generally accomplished by lowering the pH of the liquefied starch to pH 4.2 to 4.5 at 95° C. Another disadvantage of liquefaction processes is the alkaline isomerization of reducing groups. Alkaline isomerization results in the formation of a disaccharide, maltulose (4-alpha-D-glucopyranosyl-D-fructose). Maltulose lowers the glucose yield because it is resistant to hydrolysis by glucoamylases and alpha amylases. Further, it is difficult to control the formation of reversion reaction products catalyzed by active glucoamylases at high glucose concentration. Glucoamylases from Aspergillus niger are generally thermostable under the typical saccharification conditions. Therefore, a substantial amount of the glucoamylase activity may remain after the saccharification reaction. Solutions to some of the problems as discussed herein have been suggested by various researchers.
For example, Leach et al (U.S. Pat. No. 4,113,509 and U.S. Pat. No. 3,922,196) disclose a process for converting granular starch (refined) into soluble hydrolyzate by incubating the granular starch with bacterial alpha amylase at a temperature below the starch gelatinization temperature. Beta amylase was then used for hydrolysis to produce high maltose syrup.
European Patent Application No. 0350737 A2 discloses a process for producing maltose syrup by hydrolyzing a granular (purified) starch from corn, wheat, potato and sweet potato at 60° C. without the conventional liquefaction step (gelatinization followed by liquefaction at high temperature) using an alpha amylase from Bacillus stearothermophilus. 
A multi-step process to convert granular (raw) starch to glucose using a glucoamylase demonstrating raw starch hydrolyzing capability has been previously described (U.S. Pat. No. 4,618,579). However, only 60% of the starch was hydrolyzed, which then resulted in an extensive recycling process.
Not only would it be advantageous to improved upon conventional processes for granular starch conversion, but also it would be desirable to provide processes resulting in increased expression and production of the enzymes used therefore. For example, glucoamylases having granular starch hydrolyzing activity with improved characteristics such as increased specific activity, different pH ranges and/or different levels of glycosylation may be particularly advantageous for use in industrial starch conversion. The present invention not only meets some of these needs but also results in an increase in the efficiency of producing various end products obtained from starch hydrolysis.